Inhibition ofEnt-kaurene oxidation by cytokinins

Cytokinins, which have some structural similarities to ancymidol, a plant growth retardant, were tested for their effects on the cell-free oxidation ofent-kaurene. Results indicate that several cytokinins inhibit this reaction in microsomal extracts of liquid endosperm from immature wild cucumber seeds. N⁶-cyclohexanemethyladenine was the most active (inhibiting 50% of the controlent-kaurene oxidation at 2×10^–6 M). N⁶-isoamyladenine, N⁶-benzyladenine, N⁶-(²-isopentenyl)adenine and dihydrozeatin were active at successively higher concentrations. Zeatin, kinetin, adenine, N⁶-benzyladenosine, and N⁶-(isopentenyl)adenosine were inactive in this system.The basis for the inhibition ofent-kaurene oxidation by cytokinins may be similar to that of ancymidol: interaction with cytochrome P-450. A binding spectrum similar to that of ancymidol with cytochrome P-450 from wild cucumber endosperm microsomes was obtained with four active cytokinins. The cytokinin binding properties of this protein are currently under investigation.No metabolism of N⁶-benzyladenine could be detected under conditions in which the cytokinin inhibited the oxidation ofent-kaurene toent-kaurenol.This work was supported in part by National Science Foundation Grant PCM 7619279 and PCM 8016237, and a grant from Eli Lilly and Company.A portion of this work was presented as a poster paper at the Tenth International Conference on Plant Growth Substances, Madison, Wisconsin, USA, July 22–26, 1979.     

The effect of growth retardants on phytochrome-induced lettuce seed germination

Experiments were carried out to explore the involvement of the plant hormone gibberellin (GA) in the light-induced germination of lettuce seeds. Three growth retardants known to be inhibitors of GA biosynthesis were tested for their effect on red-light-induced germination. Chlormequat chloride (CCC) and AMO-1618 had no effect, but ancymidol was strongly inhibitory. Moreover, the inhibition caused by ancymidol was completely overcome by GA₃. CCC and AMO-1618 inhibit the formation ofent-kaurene, while ancymidol blocks the oxidation ofent-kaurene toent-kaurenoic acid. Ancymidol also was found to inhibit GA-induced dark germination of lettuce seeds, and this inhibition was partially reversed by higher levels of GA. Therefore, the results suggest two possibilities for the relationship between phytochrome and GA in this system: first, the rate-limiting step in the germination of light-sensitive lettuce seeds, that which is regulated by phytochrome, is the oxidation ofent-kaurene toent-kaurenoic acid. Alternatively, red-light treatment may result in the release of active GAlike substances which, in turn, induce germination. In either case the results presented here support the view that phytochrome exerts its effect on lettuce seed germination by means of GA rather than via an independent pathway.     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^?8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^?8 M and 7×10^?7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^?8 M and 6×10^?8 M, respectively.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

The biosynthesis of the gibberellin precursorent-kaurene in cell-free extracts and the endogenous gibberellins of Japanese morning glory in relation to seed development

The endogenous levels of gibberellins (GAs) determined by a combined HPLC-bioassay procedure and the formation ofent-kaurene, an immediate GA precursor, in cell-free extracts were studied in relation to seed development inPharbitis nil Choisy cv. Violet. Three biologically active GA fractions were obtained, tentatively identified as GA₃, GA₅/ GA₂₀, and a GA fraction, possibly GA₁₉ and/or GA₄₄, which all increased in activity during early seed development and subsequently declined during maturation of the seeds. The total endogenous GA level reached its maximum at 19 days after anthesis, just before the seeds had attained their maximum fresh weight at about 23 days after anthesis. Similarly, theent-kaurene synthesizing capacity showed a rapid increase during the period of rapid growth of the seeds, followed by a decline during maturation. A direct relationship between the endogenous GA levels and theent-kaurene synthesizing capacity of a particular tissue was indicated.     

Cytokinin inhibition of Arabidopsis root growth: An examination of genotype, cytokinin activity, and N6-benzyladenine metabolism

The effects of cytokinins on the in vitro growth of the roots of Arabidopsis thaliana seedlings were examined. Root growth was inhibited in a manner dependent upon the type of cytokinin compound, the cytokinin concentration, the Arabidopsis genotype, and the duration of exposure to cytokinin. For the cytokinins N ⁶-benzyladenine (BA), isopentenyl adenine (iP), or dihydrozeatin (DHZ), the concentration required for 50% root growth inhibition differed for each cytokinin and in each of three Arabidopsis genotypes tested. iP was the most active cytokinin in inhibiting the root growth of the Ler-0 genotype, whereas iP and BA had equal activity when tested with the Col-2 and Columbia genotypes. DHZ had the lowest activity of the three cytokinins tested in all three genotypes. A brief 1-day exposure of seeds to a root-inhibiting concentration of BA increased root growth compared with seedlings grown without BA; exposure to BA for 3–6 days inhibited root growth. BA metabolism was evaluated after 6 h and 1, 3, and 6 days in Columbia seedlings. BA, N ⁶-benzyladenosine (BAR), and N ⁶-benzyladenosine-5-monophosphate (BAMP) decreased with time, whereas N ⁶-benzyladenine-7--d-glucopyranoside (BA-7-G) and N ⁶-benzyladenine-9--d-glucopyranoside (BA-9-G) accumulated in the growing seedlings. Seven aromatic cytokinins were compared at 5 m for their effect on Col-3 root growth. BA, BAR, N ⁶-(m-hydroxybenzylamino)adenine, and N ⁶-(o-hydroxybenzylamino)adenine were highly effective in inhibiting root growth, whereas N ⁶-(p-hydroxybenzylamino)adenine produced only a slight decrease in root growth. BA-7-G and BA-9-G did not affect root growth.Abbreviations BA N ⁶-benzyladenine - iP isopentenyl-adenine - DHZ dihydrozeatin - BAR N ⁶-benzyladenosine - BAMP N ⁶-benzyladenosine 5-monophosphate - BA-7-G N ⁶-benzyladenine-7--d-glucopyranoside - BA-9-G N ⁶-benzyladenine-9--d-glucopyranoside - m-OH BA N ⁶-(m-hydroxybenzylamino)adenine - o-OH BA N ⁶-(o-hydroxybenzylamino)adenine - p-OH BA N ⁶-(p-hyrdoxybenzylamino)adenine - HPLC high performance liquid chromatography - gFW grams fresh weight     

Studies on the Specificity and Site of Action of alpha-Cyclopropyl-alpha-[p-methoxyphenyl]-5-pyrimidine Methyl Alcohol (Ancymidol), a Plant Growth Regulator

α-Cyclopropyl-α-[p-methoxyphenyl]-5-pyrimidine methyl alcohol (ancymidol) is an inhibitor of ent-kaur-16-ene oxidation in microsomal preparations from the liquid endosperm of immature Marah macrocarpus seeds. The K_i for this inhibitor is about 2 × 10⁻⁹ m. Ancymidol also blocks ent-kaur-16-en-19-ol and ent-kaur-16-en-19-al oxidation by the same preparations with a similar efficiency, but does not significantly inhibit ent-kaur-16-en-19-oic acid oxidation. Ancymidol appears to be specific for this series of oxidations in higher plant tissues. It does not inhibit the oxidation of kaurene nor kaurenoic acid in rat liver microsomes and has no significant effect on the oxidation of cinnamic acid in microsomal preparations from Sorghum bicolor seedlings. Ancymidol also does not inhibit kaurene oxidation in vitro nor in vivo in cultures of the fungus Fusarium moniliforme. The presence of ancymidol did not significantly alter the activities of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, or NADH-cytochrome b₅ reductase. The addition of ancymidol to suspensions of oxidized M. macrocarpus endosperm led to a difference spectrum with an absorption maximum at 427 nm and a minimum at 410 nm.     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.     

Presence of endogenous ent-kaurene in a microsomal preparation from Cucurbita maxima L. Endosperm and implications for kinetic studies of ent-kaurene oxidase

Microsomal and soluble cell-free extracts prepared from liquid endosperm of Cucurbita maxima L. were found to contain high concentrations of endogenous ent-kaurene and ent-kaurenol by gas chromatography-mass spectrometry-chemical ionization with deuterated internal standards. Increases in the levels of ent-kaurenol, ent-kaurenoic acid, and ent-7-hydroxykaurenoic acid are correlated with a decline in the amount of endogenous ent-kaurene following a 10 min incubation of microsomes with NADPH and FAD. The rate of oxidation of radiolabeled ent-kaurene by the microsomal fraction was determined, and the need to account for endogenous substrate is shown. Endogenous ent-kaurene present in soluble extracts had the effect of diluting the [¹⁴C]ent-kaurene synthesized from [¹⁴C]mevalonic acid, resulting in reduced specific radioactivity of the product. The dilution of [¹⁴C]ent-kaurene was more pronounced in extracts with higher endogenous ent-kaurene levels or when the reactions were run in the presence of O₂ and NADPH. Evidence is presented that suggests differential metabolism of endogenous ent-kaurene and radiolabeled ent-kaurene in both microsomal and soluble extracts.Abbreviations Kaurene ent-kaur-16-ene - MVA mevalonic acid - kaurenol ent-kaur-16-en-19-ol - kaurenoic acid ent-kaur-16-en-19-oic acid - EtOAc ethyl acetate - MeOH methanol - GC-MS-CI gas chromatography-mass spectrometry-chemical ionization - 13-OH KA ent-13-hydroxykaur-16-en-19-oic acid - 7-OH kaurenoic acid ent-7-hydroxykaur-16-en-19-oic acid - kaurenal ent-kaur-16-en-19-al - Me(x) methyl ester of x - TMS(x) trimethylsilyl ether or ester of x - GA(x) gibberellin A(x)     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Metabolism of cytokinin: Phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ

Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 ± 500) phosphoribosylates the bases at an optimum Mg²⁺ concentration of 5 mm and optimum pH of 7.5 (50 mm Tris-HCl buffer). K_m values for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, N⁶-benzyladenine, and adenine are 130, 110, 154, and 74 μm, respectively, in 50 mm Tris-HCl buffer (pH 7.5) at 37 °C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”     

Inhibition of gibberellin biosynthesis by paclobutrazol in cell-free homogenates ofCucurbita maxima endosperm andMalus pumila embryos

The plant growth retardant paclobutrazol, (PP333) (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursorent-kaurene toent-kaurenoic acid in a cell-free system fromCucurbita maxima endosperm. The KI₅₀ for this inhibition is 2×10^–8 M. The KI₅₀ values for the separated2S, 3S, and2R, 3R enantiomers of paclobutrazol in this system are 2×10^–8 M and 7×10^–7 M, respectively. A cell-free preparation from immatureMalus pumila embryos convertsent-kaurene to gibberellin A₉, whereas no conversion occurs in a similar preparation fromMalus endosperm. The conversion ofent-kaurene by the embryo preparation is inhibited by paclobutrazol with KI₅₀ values for the2S,3S and2R,3R enantiomers of 2×10^–8 M and 6×10^–8 M, respectively.     

Inhibition of dopamine-β-hydroxylase by cytokinins

The activity of cell-free preparations of dopamine--hydroxylase from a mammalian source was inhibited by a number of N⁶-substituted adenine derivatives that are hormonally active as cytokinins in plant systems. The synthetic cytokinin N⁶-cyclohexylmethyladenine exhibited inhibitory activity equivalent to that of 1-phenyl-3-(2-thiazolyl)-2-thiourea (PTTU), a compound known to be a potent inhibitor of dopamine--hydroxylase activity. PTTU itself was found to exhibit cytokinin activity in the tobacco callus bioassay and to inhibit the activity of the plant enzyme, cytokinin oxidase. The possible significance of these observations is discussed in relation to known effects of cytokinins on phenethylamine metabolism.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Changes in cytokinin activities before,during and after floral initiation in Polianthes tuberosa

The contents of endogenous cytokinin in tuberose corms (Polianthes tuberosa) at vegetative, early floral initiation, and flower development stages were investigated. We also determined the influence of exogenous cytokinin treatment on the corm apex at three different growth stages in relation to floral initiation and development in tuberose. The exogenous cytokinin effectively induced floral initiation and development, especially at the early floral initiation and flower development stages. Endogenous cytokinins were higher in early floral initiation and development stages in comparison to the vegetative stage. During floral initiation stage, the zeatin and dihydrozeatin increased significantly, while the cytokinins, zeatin riboside, dihydrozeatin riboside, ⁶N-(δ²-isopentenyl) adenine, and ⁶N-(δ²-isopentenyl) adenine riboside at consistently low levels. The increase of cytokinin levels in tuberose corms during floral induction suggests a role for cytokinins in tuberose apex evocation. Moreover, these results indicate that cytokinins seem to promote the development of flower buds rather than inducing flowering in tuberose.     

Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: Heterologous expression,purification and properties

The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N⁶-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q₀). In slightly acidic conditions (pH 5.0), N⁶-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N⁶-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N⁶-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.     

Photoreduction of Sulfite by Spinach Leaf Preparations in the Presence of Grana System

The β-anomer of N⁶-benzyladenosine has been long known as an artificial cytokinin.¹⁾ Recently, however, this compound was isolated from a cytokinin-autotrophic cell culture of anise, Pimpinella anisum L.²⁾ Thus, this compound now constitutes the third member of the naturally-occurring cytokinins possessing an N⁶-benzyladenine structure, together with N⁶-(O-hydroxybenzyl)adenosine isolated from leaves of Poplus robusta³⁾ and N⁶-(o-hy-droxybenzyl)-2-methylthio-9-β-d-glucofuranosyl-adenine isolated from fruits of Zantedeschia aethio-pica.⁴⁾ No information has been available concerning whether or not the a-anomer of N⁶-benzyladenosine possesses cytokinin activity.

We report here the synthesis and cytokinin activity of N⁶-benzyl-α-adenosine (N⁶-benzyl-9-α-d-ribofuranosyl adenine).